The effect of short days or timed melatonin treatments on the number and neuroanatomical location of gonadotropin-releasing hormone (GnRH) neurons was studied in the brain of the pubertal male Djungarian hamster. At the beginning of the rapid phase of testicular growth and onset of peak gonadotropin secretion (15 days of age), males were treated for 10 days with either short days (10 L:14 D; n = 6), or remained in long days (16 L:8 D; n = 5) and injected each afternoon with melatonin. These treatments arrested testicular growth compared to the gonadal development that occurred in long-day controls (n = 9). Every brain section (60 µm) from the olfactory bulb to the anterior hypothalamus was processed for GnRH immunocytochemistry and viewed under brightfield light microscopy. GnRH cell bodies had smooth contours and were morphologically bipolar or unipolar. The number of bipolar neurons was similar regardless of treatment (about 170/brain). However, fewer unipolar GnRH cell bodies (p < 0.05) were found in males in short days (73 ± 11) or in males administered melatonin (72 ± 14) compared to the unipolar number in hamsters in long days (132 ± 14). With respect to neuroanatomical distribution, significantly fewer unipolar GnRH neurons were found in the medial preoptic area of males treated with short days or melatonin (55–70% decrease) compared to cell numbers in long-day controls. The melatonin-treated hamsters also had reduced numbers of unipolar GnRH neurons in the diagonal band of Broca relative to the number of unipolar neurons in long-day controls. In the locations lateral and caudal to these two regions, the number of GnRH somata were not significantly different among the three treatment groups. The present study implicates the medial preoptic area and, possibly, the diagonal band of Broca as involved in the photoperiodic control of reproductive development. Moreover, the association of fewer morphologically unipolar GnRH neurons in these areas with delayed puberty, as induced by short day or melatonin treatment, suggests the involvement of this subtype in the normal process of sexual maturation.